This application is based upon the conviction that activation of factor IX (FIX) by the factor VIIa/tissue factor (FVIIa/TF) complex and by factor XIa (FXIa) represents key steps in the initiation and sustained phases of coagulation, respectively. All of the specific aims represent continuation of our ongoing studies. FIX consists of an N-terminal Gla domain followed by two EGF-like domains, an activation peptide (AP) segment, and a C- terminal serine protease domain. In specific aim 1, we will determine the specific residues in the Gla domain of FIX/FIXa that interact with the Apple 3 domain of FXIa using mutagenesis, kinetic, and direct binding studies. To validate our biochemical data, we will crystallize active site inhibited FIXa with the isolated Apple 3 domain of FXIa and solve its structure. A second part of this aim involves investigations of the molecular basis of hemophilia C resulting from CRM+ missense mutations at or near the active site of FXIa. In specific aim 2, we will determine the specific residues in the Gla and EGF1 domain of FIX/IXa that interact with FVIIa/TF using mutagenesis, kinetic, and direct binding studies. We have recently obtained crystals of FVIIa/soluble TF that diffract to 1.6 E, the highest resolution data obtained thus far. The structure reveals functional TF165/166 residue region implicated in binding to FIX/IXa but was disordered in previous structures. To validate the implicated binding region and the biochemical data, we now propose to crystallize chimeric FXa molecule containing the Gla/EGF1 domains of FIX in complex with two-domain TFPI/FVIIa/soluble TF and solve its structure. In specific aim 3, we will first refine the structure of the EGF2-Protease domain segment of FIXa using the 1.67 E diffraction data that we have obtained. We now propose to co-crystallize the EGF2-Protease (E2P) domain segment of FIXa with the isolated A2 domain of FVIIIa. This structure should verify the interface region between the A2 domain of FVIIIa and the EGF2-Protease domain of FIXa. We will also continue our investigations toward identifying a new antithrombotic lead candidate molecule that inhibits FIXa:FVIIIa interaction at the interface between the FVIIIa A2 domain and the FIXa EGF2-Protease domain. A promising small molecule, peptidyl or nonpeptidylmimetics, will be tested for antithrombotic potential in both tail bleeding as well as carotid artery mouse models. In vitro assays will be performed to determine the selectivity and specificity of such molecules for FIXa:FVIIIa inhibition. The proposed studies have the potential to develop new generation of antithrombotics. PUBLIC HEALTH RELEVANCE: Multiple proteins interact with each other to initiate and propagate blood coagulation in mammals including humans. Two proteins that are missing in hemophilia are factors IX and VIII. This project aims to understand how these two proteins interact with each other and what is the molecular basis of hemophilia? Using this knowledge, we are developing antithrombotics that would disrupt this interaction and prevent heart attacks and thrombosis.